Electrophoresis is a technique used to separate charged species on the basis of size, electric charge, and other physical properties. In electrophoresis, the charged species migrate through a conductive electrophoretic medium, which may be (but is not required to be) a gel, under the influence of an electric field. Activated electrodes located at either end of the electrophoretic medium provide the driving force for the migration. The properties of the molecules, including their charge and mass, determine how rapidly the electric field causes them to migrate through the electrophoretic medium.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionizable groups. Because of these ionizable groups, at any given pH, many important biological molecules exist in solution as electrically charged species. The electrically charged species enable doctors and scientists to separate nucleic acids and proteins using electrophoresis.
Separation of molecules, biological or otherwise, using electrophoresis depends on various forces, including charge and mass. When a biological sample, such as a protein or DNA, is mixed in a buffer solution and applied to an electrophoretic medium, these two forces act together. Separation using electrophoresis is possible because the rate of molecular migration through the electric field depends on the strength of the field, the charge, size, and shape of the molecules, and the ionic strength and temperature of the buffer through which the molecules are moving. During electrophoresis, the applied electrical field causes the molecules to move through the pores of the electrophoretic medium based on the molecular charge. The electrical potential at one electrode repels the molecules while the potential at the other electrode simultaneously attracts the molecules. The frictional force of the electrophoretic medium also aids in separating the molecules by size. Typically, after the applied electrical field has been removed, the molecules may be stained. After staining, the separated macromolecules can be seen in a series of bands spread from one end of the electrophoretic medium to the other. If these bands are sufficiently distinct, the molecules in these zones can be examined and studied separately by fixing macromolecules and washing the electrophoretic medium to remove the buffer solution.
Casting of electrophoresis gels, e.g. those of polyacrylamide or agarose, is commonly done by creating a series of sample wells in the gel surface. Liquid mixtures to be analyzed are loaded into the well, typically using a pipette, syringe needle, electrophoretic comb, or similar sample delivering instrument. However, intra-sample band resolutions are only as good as the width of the sample applied, and because small sample volumes are subject to surface tension (establishing micelle diameters in excess of the resolution desired for loading sample in a particular well) such band resolutions are poor and variable. In addition, microvolumes are often applied in a 2-D-like approach which prevents volumetric applications. Further, sample kinetics within the gel are limited relative to free solution chemistries; compromised degrees of freedom negatively affect uniform product or adduct development, except in localized areas that are often only a few sample lanes in dimension. For these reasons, sample reproducibility at the 1 uL level is often too imprecise to be clinically/analytically acceptable.
In addition, there is great difficulty in determining the concentration of fractions of a test sample separated within an electrophoretic matrix without use of an external method. Attempts to incorporate such a reference internal standard within an electrophoretic assay have been unsuccessful. Traditional attempts at polyvalent antiseras and multiple proteins failed due to variable avidity and dye binding characteristics. Qualitative markers of identity and positional references are available, but none use an internal reference standard that would allow concentrations for electrophoretically separated fractions to be absolutely quantitated independent of external methods.
There is no predicate technology in this area. Electrophoresis has been and remains the analytic derivative of “total” chemistries, where all components of a sample undergo the same processing. Components are not removed by precipitation, capture, or other method; they are separated on the same substrate and can be recombined by reversing the process. This fact has limited the capacity of electrophoresis to generate absolute quantity values. Conventional electrophoresis measures relative concentrations, i.e., it calculates percentage fractions as the area under curves from detected bands that have been translated into signals to produce electropherograms. In particular, after electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry. An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. For example, if the electrophoresis is being used for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration; if the electrophoresis is being used for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity.
Thus, conventional electrophoresis can only assign relative percentage values for all bands detected within a sample. For example, if multiple bands are developed for cholesterol, relative percentages are provided by a densitometer and an externally-provided total cholesterol value is proportionately distributed amongst the fractions to determine absolute concentrations. For example, two bands are detected by densitometer—the first is 25% and the second 75% of the total signal detected. Given a total analyte concentration of 200 mg/mL, the first band is 50 mg/mL, (0.25×200 mg/mL), the second is 150 mg/mL, (0.75×200 mg/mL). No method exists to provide calibrators on a gel and/or within each individual sample.
Because electrophoresed patterns are only as useful as their resolutions, and due to the problems noted above, a great need exists for a method and/or system that removes the inter-sample variability and intra-sample errors associated with gel electrophoresis technology.
This invention is directed to overcoming these and other deficiencies in the art.